The purpose of the proposed research is to strengthen the foundation for the rational development of drug-like small-molecule compounds with potential therapeutic value in invasive/metastatic cancers and inflammatory disorders by advancing the understanding of the CD44:hyaluronan protein:carbohydrate binding interaction. This binding interaction, which is a validated target for anti-cancer and anti-inflammatory therapy, has been the subject of both NMR and X-ray crystallography structural biology studies that have led to atomic-resolution models. However, conformational flexibility of the binding site, a large protein:carbohydrate binding interface, and an order-to-disorder transition at an allosteric site are all factors that complicate attempts to understand binding at an atomic level of detail. The result is a critical barrier to progress both with respect to understanding the molecular basis for CD44:hyaluronan molecular recognition and the structure-based discovery of drug-like inhibitors of CD44:hyaluronan binding. Toward overcoming this barrier, atomically- detailed molecular simulations will be applied to achieve three specific aims: 1. Determining the contribution of binding site flexibility to binding affinity, 2. Looking for "hot spots" on the large binding interface that make greater than expected contributions to binding affinity, and 3. Determining the contribution of the allosteric site order-to-disorder transition on binding site flexibility and affinity. The research design involves systematically determining the energetics of the conformational landscape underlying weak and dynamic protein:carbohydrate interactions, the role of which has not been explored extensively via either experimental or computational approaches. The methodology to be used is all-atom explicit-solvent molecular dynamics simulations, including biased sampling and computational alchemy approaches that provide information regarding the free-energy consequences of conformational and compositional changes in the CD44:hyaluronan complex. Achieving the stated aims will lead to atomic- resolution understandings of (a) binding site flexibility and affinity, and (b) how flexibility and affinity are impacted by the order-to-disorder transition, thereby furthering the fundamental understanding of CD44:hyaluronan molecular recognition and its application to structure-based discovery of CD44:HA inhibitors.) PUBLIC HEALTH RELEVANCE: The binding interaction of the CD44 protein and the hyaluronan carbohydrate plays roles in certain invasive/metastatic cancers and inflammatory disorders. Targeting this interaction may therefore be a new way to help treat these diseases. Toward this goal of new therapeutics, and also to enhance the limited understanding of molecular flexibility in protein:carbohydrate complexes, the present work seeks to strengthen the fundamental molecular-level understanding of this binding interaction through the application of cutting- edge computer simulation methods.